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The American bullfrog (Lithobates catesbeianus [Shaw, 1802]) is one of the hundred most destructive Alien Invasive Species (AIS) worldwide that has invaded more than 40 countries across 4 continents. In Belgium, bullfrogs have occupied a large area in a relatively short period of time despite a decade of intensive management interventions. Acquiring better insights into the distribution, abundance, and spatial spread of this invasive frog species is an important first step towards a successful management strategy. In this study, we sampled 382 permanent water bodies and combined environmental DNA (eDNA)-based analyses using quantitative droplet digital PCR (ddPCR) with assessments of habitat characteristics to generate an overview of the present distribution of bullfrogs in Flanders (Northern Region of Belgium) and the type of water bodies they have invaded. Our results revealed a fragmented distribution pattern covering an area of 364.76 km² that consisted of eight metapopulations located in five different river valleys, suggesting the occurrence of multiple anthropogenically-mediated introductions. Bullfrogs appeared to be firmly established in the valley of the Grote Nete, where invaded waterbodies have been found along 72% of the length of this river, divided into three distinct metapopulations. Unlike refuge sites, bullfrogs were found to be highly selective in their choice of breeding sites, which were characterised by abundant emergent vegetation and sparse tree cover along the shoreline. The division of the vast occupied area into well-defined, accurately delineated metapopulations facilitates the identification of functional management units. Furthermore, the obtained knowledge of the patterns of range expansions and the spatial configuration and associated environmental features of breeding sites can be used to prioritise management interventions in strategically located invasion hubs. Overall, we conclude that eDNA-based monitoring combined with environmental assessments provide important information that can be used to manage widespread aquatic AIS more effectively. ispartof: Management Of Biological Invasions vol:14 status: Published online

Monitoring fish communities is central to the evaluation of ecological health of rivers. Both presence/absence of fish species and their relative quantity in local fish assemblages are crucial parameters to measure. Fish communities in lotic systems are traditionally monitored via electrofishing, characterized by a known limited efficiency and high survey costs. Analysis of environmental DNA could serve as a non-destructive alternative for detection and quantification of lotic fish communities, but this approach still requires further insights in practical sampling schemes incorporating transport and dilution of the eDNA particles; optimization of predictive power and quality assurance of the molecular detection method. Via a controlled cage experiment, we aim to extend the knowledge on streamreach of eDNA in small rivers and large brooks, as laid out in the European Water Framework Directive's water typology. Using a high and low source biomass in two river transects of a species-poor river characterized by contrasting river discharge rates, we found strong and significant correlations between the eDNA relative species abundances and the relative biomass per species in the cage community. Despite a decreasing correlation over distance, the underlying community composition remained stable from 25 to 300 m, or up to 1 km downstream of the eDNA source, depending on the river discharge rate. Such decrease in similarity between relative source biomass and the corresponding eDNA-based community profile with increasing distance downstream from the source, might be attributed to variation in species-specific eDNA persistence. Our findings offer crucial insights on eDNA behaviour and characterization of riverine fish communities. We conclude that water sampled from a relatively small river offers an adequate eDNA snapshot of the total fish community in the 300-1000 m upstream transect. The potential application for other river systems is further discussed. ispartof: ENVIRONMENTAL RESEARCH vol:228 ispartof: location:Netherlands status: accepted

To effectively monitor, manage and protect aquatic species and understand their interactions, knowledge of their spatiotemporal distribution is needed. In this study, we used a fine‐scale spatiotemporal water sampling design, followed by environmental DNA (eDNA) 12S metabarcoding, to investigate occupancy patterns of a natural community of fish and amphibian species in a lentic system. In the same system, we experimentally estimated the spatial and temporal dispersion of eDNA by placing a community of different fish and amphibian species in cages at one side of the pond, creating a controlled point of eDNA emission. Analyses of this cage community revealed a sharp spatial decline in detection rates and relative eDNA quantities at a distance of 5–10 m from the source, depending on the species and its abundance. In addition, none of the caged species could be detected 1 week after removal from the system. This indicates high eDNA decay rates and limited spatial eDNA dispersal, facilitating high local resolution for monitoring spatial occupancy patterns of aquatic species. Remarkably, for seven of the nine cage species, the presence of a single individual could be detected by pooling water of subsamples taken across the whole water body, illustrating the high sensitivity of the eDNA sampling and detection method applied. Finally, our work demonstrated that a fine‐scale sampling design in combination with eDNA metabarcoding can cover total biodiversity very precisely and allows the construction of consistent spatiotemporal patterns of relative abundance and local distribution of free‐living fish and amphibian species in a lentic ecosystem.

Isogenus nubecula is a critically endangered Plecoptera species. Considered extinct in the UK, I. nubecula was recently rediscovered (in one location of the River Dee, Wales), after 22 years of absence. In a similar way to many other species of Perlodidae, I. nubecula could be utilised as a bio-indicator, for assessing water quality and health status of a given freshwater system. However, conventional monitoring of invertebrates via kick-sampling, is invasive and expensive (time consuming). Further, such methods require a high level of taxonomic expertise. Here, we compared the traditional kick-sampling method with the use of eDNA detection using qPCR and ddPCR-analyses. In spring 2018, we sampled eDNA from twelve locations on the River Dee. I. nubecula was detected using kick-sampling in five of these locations, three locations using both eDNA detection and kick-sampling and one location using eDNA detection alone – resulting in a total of six known and distinct populations of this critically endangered species. Interestingly, despite the eDNA assay being validated in vitro and in silico, and results indicating high sensitivity, qPCR analysis of the eDNA samples proved to be ineffective. In contrast, ddPCR analyses resulted in a clear detection of I. nubecula at four locations suggesting that inhibition most likely explains the large discrepancy between the obtained qPCR and ddPCR results. It is therefore important to explore inhibition effects on any new eDNA assay. We also highlight that ddPCR may well be the best option for the detection of aquatic organisms which are either rare or likely to shed low levels of eDNA into their environment.

Monitoring of fish assemblages in the Belgian part of the North Sea (BPNS) mainly happens through trawling. While effective, this method is invasive and destructive as it disturbs bottom communities, catches non-target species and removes organisms from the environment. A more sustainable alternative for monitoring marine diversity is the use of environmental DNA (eDNA) which comprises intra- and extracellular genetic material that comes from the shedding of organic material, like scales and mucus in the case of fish. When applying metabarcoding on eDNA, community composition can be inferred simply by analysing a small volume of water. Therefore the technique does not disturb the environment, and the high sensitivity of eDNA allows the detection of rare and transient species that are frequently missed by traditional sampling methods. Next to determining community composition, the amount of eDNA copies in the water could potentially be used to quantify target fish species in the marine environment. Here, we investigate whether eDNA concentrations from marine water samples correlate with local fish abundance estimates obtained via traditional beam trawling. Species specific Droplet Digital Polymerase Chain Reaction (ddPCR) assays were designed and tested for three economically important species: common sole (Solea solea), plaice (Pleuronectes platessa) and whiting (Merlangius merlangus). In march 2020, 12 sites in the BPNS were selected based on absence, low and high abundances of the three target species as observed in epibenthos monitoring data from previous years. In each site, 2L of seawater was collected with a niskin bottle from ca 1m above the sea floor. Subsequently, beam trawl transects of 1 km were conducted and all epibenthos species caught in the trawling net were morphologically identified, counted and weighted. Our results indicate promising correlations between eDNA concentrations obtained with the ddPCR assays and the number of specimens in the net for all three species. Some “false” positive results were obtained with the ddPCR, but these may actually be “true” positive detections because the fish might be present in the area but were not caught in the trawling transects. This warrants further investigation to see how far eDNA signals can be detected in the North Sea system. Next, 50 water samples were collected in Autumn 2020, involving more locations with or without the three fishes. This time samples were taken at the beginning, middle and end of the 1 km transects to investigate small scale horizontal variation in eDNA concentrations. The autumn samples are currently being processed. In March 2021, samples will be taken at different depths (surface, middle of the water column and ca 1m above the seafloor) to investigate whether there are any vertical patterns in eDNA distribution in a very well mixed system such as the BPNS. A DNA shedding experiment will be performed as well to estimate the rate at which the three fishes shed DNA. This will provide important information on how quickly fishes can be detected when they swim by. The information obtained with the field sampling and experimental setting will help to strengthen the correlation to a point that reliable abundance estimations of our target species become possible and will allow us to evaluate the potential of eDNA as a sustainable alternative/addition to traditional monitoring methods.

In recent years, environmental DNA analyses became increasingly integrated to detect and monitor the presence and abundance of rare organisms, especially in inaccessible aquatic habitats. Although it is generally proven that detection probabilities of eDNA surveys exceed those obtained via conventional techniques, these molecular approaches are, however, also subjected to detection limitations and levels of uncertainty. Besides improvements that can be made in terms of sampling design, volumes of filtered water, and the effective quantity of DNA that is finally analysed, the sensitivity of eDNA surveys is inherently determined by the number of target eDNA copies suspended in the water column. Here we show that multiplexing different primer/probe assays for the same species, but targeting amplicons situated at different loci, is a surprisingly overlooked aspect that can substantially contribute to reduce these limitations and increase the sensitivity of single-species detections. By empirically testing a large number of natural eDNA samples via ddPCR, we reveal that the use of multiple markers can significantly lower the LOD and LOQ of rare and elusive species, such as the invasive American bullfrog and the endangered European weather loach in a variety of different water bodies, such as ponds, lakes, streams, canals, etc. Especially at very low eDNA concentrations of both target species, our results showed that analysing mulitple loci significantly increased detection probabilities and lowered stochasticity effects, and thus ultimately reduces PCR costs when analysed in multiplex. The validation and use of more than one assay taregtting a single species, may further increase the confidence of positive detections. Finally, we illustrate that the implementation of internal positive controls (IPC's), is an absolute must for accurate validation of eDNA workflows and reliable interpretation of the generated data. IPC’s not only help to track down degraded and inhibited samples, to avoid false-negative detections, it also offers insights into extraction efficiency, indispensable for accurate quantification of population densities. Overall, our findings provide strong support that the multiplexing of multiple markers on different loci in combination with the use of internal positive controls ensures increased detection rates at very low eDNA concentrations and generates more robust and reliable data.

In 2020, four populations of the marbled crayfish Procambarus virginalis, which is included on the list of Invasive Alien Species of Union concern, were discovered in northern Belgium. These records represent the first established populations of this invasive parthenogenetic species in the Benelux. The marbled crayfish seems wellestablished at all sites where it was discovered. Genetic analysis confirmed the species’ identity with the obtained COI Folmer fragments being 100 percent identical to reference sequences of P. virginalis from Germany, Italy, Sweden and the Czech Republic. We proposed a single diagnostic nucleotide for unambiguous character-based species identification between P. virginalis and P. fallax. The finding of this new species through opportunistic surveys instigated by citizen science reports indicates considerable knowledge gaps on crayfish distribution in Belgium. Considering the regulated status of most species in Belgium, we advocated the further set-up of dedicated crayfish surveillance using passive and active monitoring including environmental DNA detection. ispartof: Bioinvasions Records vol:10 issue:2 pages:326-340 status: published

ExoticPopulustaxa pose a threat to the success of riparian forest restoration in floodplain areas. We evaluated the impact of exoticPopulustaxa on softwood riparian forest development along the river Common Meuse after introducing nativePopulus nigraand after the re-establishment of the natural river dynamics. We sampled 154 poplar seedlings that spontaneously colonized restored habitat and assessed their taxonomy based on diagnostic chloroplast and nuclear microsatellite markers. Furthermore, by using a paternity analysis on 72 seedlings resulting from six open pollinatedP. nigrafemales, we investigated natural hybridization between frequently planted cultivated poplars and nativeP. nigra. The majority of the poplar seedlings from the gravel banks analyzed where identified asP. nigra; only 2% of the sampled seedlings exhibited genes of exotic poplar species. Similarly, the majority of the seedlings from the open pollinated progenies were identified asP. nigra. For three seedlings (4%), paternity was assigned to a cultivar ofP.×canadensis. Almost two decades after reintroducingP. nigra, the constitution of the seed and pollen pools changed in the study area in favor of reproduction of the native species and at the expense of the exotic poplar species. This study indicates that, although significant gene flow form exotic poplars is observed in European floodplains, restoration programs of the nativeP. nigracan vigorously outcompete the exotic gene flows and strongly reduce the impact of exoticPopulustaxa on the softwood riparian forest development.

Exotic Populus taxa pose a threat to the success of riparian forest restoration in floodplain areas. We evaluated the impact of exotic Populus taxa on softwood riparian forest development along the river Common Meuse after introducing native Populus nigra and after the re-establishment of the natural river dynamics. We sampled 154 poplar seedlings that spontaneously colonized restored habitat and assessed their taxonomy based on diagnostic chloroplast and nuclear microsatellite markers. Furthermore, by using a paternity analysis on 72 seedlings resulting from six open pollinated P. nigra females, we investigated natural hybridization between frequently planted cultivated poplars and native P. nigra. The majority of the poplar seedlings from the gravel banks analyzed where identified as P. nigra; only 2% of the sampled seedlings exhibited genes of exotic poplar species. Similarly, the majority of the seedlings from the open pollinated progenies were identified as P. nigra. For three seedlings (4%), paternity was assigned to a cultivar of P. × canadensis. Almost two decades after reintroducing P. nigra, the constitution of the seed and pollen pools changed in the study area in favor of reproduction of the native species and at the expense of the exotic poplar species. This study indicates that, although significant gene flow form exotic poplars is observed in European floodplains, restoration programs of the native P. nigra can vigorously outcompete the exotic gene flows and strongly reduce the impact of exotic Populus taxa on the softwood riparian forest development.

1.AbstractExoticPopulustaxa pose a threat to the success of riparian forest restoration in floodplain areas. We evaluated the impact of exoticPopulustaxa on softwood riparian forest development along the river Common Meuse after introducing nativePopulus nigraand after the re-establishment of the natural river dynamics. We sampled 154 poplar seedlings that spontaneously colonised restored habitat and assessed their taxonomy based on diagnostic chloroplast and nuclear microsatellite markers. Furthermore, by using a paternity analysis on 72 seedlings resulting from six open pollinatedP. nigrafemales, we investigated natural hybridization between frequently planted cultivated poplars and nativeP. nigra. The majority of the poplar seedlings from the gravel banks analyzed where identified asP. nigra; only 2% of the sampled seedlings exhibited genes of exotic poplar species. Similarly, the majority of the seedlings from the open pollinated progenies were identified asP. nigra. For three seedlings (4%), paternity was assigned to a cultivar ofP. xcanadensis. Almost two decades after reintroducingP. nigra, the constitution of the seed and pollen pools changed in the study area in favour of reproduction of the native species and at the expense of the exotic poplar species. This study indicates that, although significant gene flow form exotic poplars is observed in European floodplains, restoration programmes of the nativeP. nigracan vigorously outcompete the exotic gene flows and strongly reduce the impact of exoticPopulustaxa on the softwood riparian forest development.

The European weather loach (Misgurnus fossilis) is a cryptic and poorly known fish species of high conservation concern. The species is experiencing dramatic population collapses across its native range to the point of regional extinction. Although environmental DNA (eDNA)-based approaches offer clear advantages over conventional field methods for monitoring rare and endangered species, accurate detection and quantification remain difficult and quality assessment is often poorly incorporated. In this study, we developed and validated a novel digital droplet PCR (ddPCR) eDNA-based method for reliable detection and quantification, which allows accurate monitoring of M. fossilis across a number of habitat types. A dilution experiment under laboratory conditions allowed the definition of the limit of detection (LOD) and the limit of quantification (LOQ), which were set at concentrations of 0.07 and 0.14 copies μl-1 , respectively. A series of aquarium experiments revealed a significant and positive relationship between the number of individuals and the eDNA concentration measured. During a 3 year survey (2017-2019), we assessed 96 locations for the presence of M. fossilis in Flanders (Belgium). eDNA analyses on these samples highlighted 45% positive detections of the species. On the basis of the eDNA concentration per litre of water, only 12 sites appeared to harbour relatively dense populations. The other 31 sites gave a relatively weak positive signal that was typically situated below the LOQ. Combining sample-specific estimates of effective DNA quantity (Qe ) and conventional field sampling, we concluded that each of these weak positive sites still likely harboured the species and therefore they do not represent false positives. Further, only seven of the classified negative samples warrant additional sampling as our analyses identified a substantial risk of false-negative detections (i.e., type II errors) at these locations. Finally, we illustrated that ddPCR outcompetes conventional qPCR analyses, especially when target DNA concentrations are critically low, which could be attributed to a reduced sensitivity of ddPCR to inhibition effects, higher sample concentrations being accommodated and higher sensitivity obtained. ispartof: JOURNAL OF FISH BIOLOGY vol:98 issue:2 pages:399-414 ispartof: location:England status: published

Isogenus nubeculais a critically endangered Plecoptera species. Considered extinct in the UK, the species was recently rediscovered in one location of the river Dee in Wales after 22 years of absence. As many species belonging to the Perlodidae, this species can be a bio-indicator, utilised for assessing water quality and health status of a given freshwater system. However, conventional monitoring of invertebrates via kick-sampling for example, is an invasive and expensive (time consuming). Further, such methods require a high level of taxonomic expertise. Here, we compared the traditional kick-sampling method with the use of eDNA detection using qPCR and ddPCR-analyses. In spring 2018, we sampled eDNA from twelve locations on the river Dee.I. nubeculawas detected using kick-sampling in five of these locations, three locations using both eDNA detection and kick-sampling and one location using eDNA detection alone – resulting in a total of six known and distinct populations of this critically endangered species. Interestingly, despite the eDNA assay being validated in vitro and in silico, and results indicating high sensitivity, qPCR analysis of the eDNA samples proved to be ineffective. In contrast, ddPCR analyses resulted in a clear detection ofI. nubeculaat four locations suggesting that inhibition most likely explains the big discrepancy between the obtained qPCR and ddPCR results. It is therefore important to explore inhibition effects on any new eDNA assay. We also highlight that ddPCR may well be the best option for the detection of aquatic organisms which are either rare or likely to shed low levels of eDNA into their environment.

Short notes have no abstract

International audience; Interspecific hybrids of Populus species are known for their superior growth. In this study, we examined the effect of the genetic background and contrasting environmental conditions on growth and searched for quantitative trait loci (QTL) affecting growth traits. To this end, two hybrid poplar families resulting from controlled crosses, Populus deltoides ‘S9-2’×P. nigra ‘Ghoy’ (D×N, 180 F1) and P. deltoides ‘S9-2’×P. trichocarpa ‘V24’ (D× T, 182 F1), were grown at two contrasting sites, Northern Italy and Central France. At the end of the second growing season, tree dimensions (stem height, circumference, and volume) were assessed. The performances of both families significantly differed within and between sites. Tree volume was significantly larger at the Italian site as compared to the French site. Genotype by environment interactions were significant but low for both families and for all growth traits. Tight correlations among the individual growth traits indicated that there may be a common genetic mechanism with pleiotropic effects on these growth traits. In line with previous studies, linkage groups I, VII, IX, X, XVI, XVII, and XIX appeared to have genomic regions with the largest effects on growth traits. This study revealed that (1) both families have high potential for selection of superior poplar hybrids due to the pronounced heterosis (hybrid vigor) and the large genetic variability in terms of growth and (2) the choice of site is crucial for poplar cultivation.